Cytokine flow cytometry (CFC) is a novel technique for analysis of antigen-specific immune responses by the detection of intracellular cytokines in CO4 and/or CD8 T cells. Briefly, whole blood is incubated with specific antigen (viral lysate, purified proteins, or peptides) in the presence of co-stimulatory mAb to CD28 and CD49d. Brefeldin A is added to allow accumulation of intracellular cytokines. After a total of six hours, adherent cells are removed with EDTA, then leukocytes are fixed and erythrocytes lysed with F ACS Lysing Solution. Cells are washed and permeabilized with F ACS Permeabilizing Solution to allow intracellular staining with a 3- or 4-color mAb cocktail. Positive responses are defined as cells producing cytokine and CD69, an early activation antigen. The goal of this project will be to apply the CFC technique to the detection of T cell responses, first, to foreign proteins in the PBL of normal donors and cancer patients. In this specific aim, reference ranges for responses to CMV, flu, and KLH will be determined, and a feasible method for using cryopreserved samples will be validated. Based upon preliminary data, this will be possible using specific freezing/thawing protocols and using peptide mixes as antigens. We will produce such peptide mixes for CMV pp65 as well as for the cancer antigens in Specific Aim 2. Immune augmentation after immunization with CMV, flu, and KLH will be demonstrated using CFC, and correlations will be made with tetramer, ELISPOT, and antibody assays. In the second specific aim, the CFC technique will be applied to the detection of T cell responses to CEA, HER-2/neu, gp100, and MAGE 3 in both normal donors and cancer patients. This will establish a reference range and assay system that meets CLIA specifications. Immune augmentation after immunization will be determined by CFC, and correlations made with tetramer, ELISPOT, and antibody assays. In the third specific aim, CEA and neu-transgenic mice will be used to determine whether CFC assays can predict tumor protection after vaccination of animals with protective versus non-protective regimens. In the final specific aim, CFC assays will be applied to human clinical trials in cancer patients receiving CEA, HER-2/neu, gp100, and MAGE 3 vaccines. We will determine in this aim the feasibility of CFC analysis in institutional clinical trials, the levels of response that can be achieved, and the correlation of CFC with other assays.